1. Introduction
In recent years, interest and demand for minimally processed vegetables has increased dramatically worldwide, and leafy vegetables have achieved great economic importance in the global market. In fact, the increase in demand for vegetables over the past 25 years has led to the global value of trade in vegetables exceeding that of cereals [
1]. This trend is due to the growing interest of consumers for healthy foods [
2,
3,
4,
5], and their awareness of the role of fresh fruits and vegetables in reducing the risk of cancers as well as many other degenerative diseases [
6,
7]. Moreover, the modern frenetic lifestyle has increased the demand for healthy foods characterized by high convenience of use, which resulted in the success of minimally processed vegetables [
8,
9].
Among vegetables, ready-to-eat (RTE) products are those that are increasing most rapidly [
10], even in developing countries [
11]. Thus, the demand for raw vegetables for RTE production is also increasing, which necessitates finding new cultivation systems and techniques that can supply a sufficient amount of high-quality vegetables and enhance quality retention after minimal processing and during postharvest storage. Thus, it would be highly worthwhile to find new ways to improve the productivity and postharvest quality of vegetable crops with cost-effective and easy-to-use techniques, while at the same time limiting the impact on the environment and reducing food waste [
12,
13,
14,
15,
16].
Genetic improvement, innovative cultivation systems, and use of plant-growth regulators (PGRs) could help in reaching these goals. PGRs are used in high-value horticultural crops (e.g., tomato, eggplant, strawberry, artichoke, etc.) during cultivation, as well as during postharvest management to increase yield, enhance crop management, and improve the quality of produce [
17]. The use of PGRs, particularly gibberellins (GAs), has gained a renewed interest, as they address improper source–sink relationships and plant hormonal imbalances that may occur during growth [
18]. GAs are essential growth hormones found in plants and fungi and have several effects on plant development. They can promote stem and root elongation, leaf expansion, flowering, fruit senescence, seed germination or dormancy, and so on [
19]. Exogenous applications of GAs have actively influenced many physiological activities such as vegetative growth, flowering and flower morphology, earliness, fruit set, ion transport and osmoregulation, leaf area expansion, and internode elongation. GAs have also increased biomass production, fruit weight, and dry matter production [
20,
21,
22,
23,
24,
25,
26,
27,
28,
29], and have been used to control and slow down postharvest changes in fruits, vegetables, and flowers [
30,
31,
32,
33]. These effects are, in some cases, controversial as they can differ considerably depending on hormone demand, relative concentrations, and plant responses at various physiological and growth stages [
34]. The use of gibberellic acid (GA
3) to improve crop productivity or to control postharvest quality has been tested on many fruits and vegetables [
18,
30,
31,
32,
33,
35,
36,
37,
38,
39,
40,
41]. Nevertheless, the effects of preharvest treatment with this phytohormone on postharvest characteristics of leafy vegetables still needs to be further investigated. In a previous study, we investigated the effect of suppling low levels of GA
3 (0, 10
−8, 10
−6, and 10
−4 M GA
3) to leaf lettuce and rocket plants through the mineral nutrient solution of a hydroponic floating system and evaluated their effects on growth and quality, showing that the addition of 10
−6 M GA
3 to the nutrient solution of a hydroponic floating system can promote growth and quality of lettuce and rocket plants at harvest [
42]. The exogenous supply of GA
3 during plant growth might also exert some effects on postharvest life of vegetables that are worth investigating. Therefore, the aim of this work was to evaluate the quality characteristics during cold storage of minimally processed leaf lettuce and rocket, obtained from plants grown in a hydroponic floating system with mineral nutrient solutions containing different levels of GA
3.
2. Materials and Methods
2.1. Leafy Vegetable Cultivation
This study was carried out in greenhouse conditions at the Department of Agricultural, Food, and Forest Sciences (SAAF-University of Palermo, Palermo, Italy) (38°6′28″ N 13°21′3″ E; altitude 49 m) using a hydroponic floating cultivation system. Plants were grown under natural light conditions; the mean air temperature was 20 °C, varying between 12 and 27 °C, and relative humidity averaged 69%. A mineral nutrient solution (MNS) was used, with 4.5 mM Ca
2+, 2 mM H
2PO
4−, 1.25 mM NH
4+, 1 mM Mg
2+, 19 mM NO
3−, 11 mM K
+, 1.1 mM SO
42−, 40 μM Fe
3+, 5 μM Mn
2+, 4 μM Zn
2+, 30 μM BO
33−, 0.75 μM Cu
2+, and 0.50 µM Mo [
43] in tap water (electrical conductivity (EC) 480 μS cm
−1; pH 7.7). MNS was then mixed with gibberellic acid (Gibrelex, Biolchim, Bologna, Italy), selected from a previous study [
42], to create three concentrations: 0, 10
−8, and 10
−6 M GA
3. The EC of the MNS was 2.25 mS cm
−1, and the pH was 5.8.
Plantlets with 3–4 true leaves of leaf lettuce (
Lactuca sativa L. var.
Crispa cv. ‘Lattuga da Taglio a Foglia Liscia’, Sementi Dotto—SDD SPA, Udine, Italy) and rocket (
Eruca sativa L. cv. ‘Coltivata da orto’, Sementi Dotto—SDD SPA, Udine, Italy), were transplanted (13 March) into drilled polystyrene panels (400 plants m
−2) that were then placed to float in tubs (100 cm long × 50 cm wide × 15 cm deep, containing 75 L of treatment solution). Each treatment consisted of four replicated tubs for each GA
3 concentration (12 tubs for each leafy vegetable). Leaf lettuce and rocket have a short crop cycle, so it was not necessary to aerate the nutrient solutions during plant growth [
44]. The MNS was monitored every day for water consumption and every week for EC and pH. The tubs were replenished with new MNS with the same GA
3 concentration when the volume of MNS inside them dropped by 20%.
At harvest (21 d after transplanting), all plants were collected, and marketable yield was calculated.
2.2. Minimal Processing and Cold Storage
Soon after harvesting, plants were transferred to the laboratory of Vegetable Analysis of the SAAF Department and immediately processed, using common practices for fresh-cut production. Leaves were removed from stalks, and those with defects, yellowing, or decay were discarded. Then, they were washed twice with tap water for 5 min, and dried for 1 min with a handheld salad spinner by manual centrifugation. At the end of processing, the yield of minimally processed product was determined.
Samples of 100 g of each GA3 treatment and each leafy vegetable were immediately packed in PE bags, sealed with a hot bar (Laica VT3112, Vicenza, Italy), and kept at 4 °C for 21 d. At the end of processing and after 7, 14, and 21 d of cold storage, the physicochemical characteristics of randomly selected samples were assessed. Weight loss was estimated by weighing samples soon after processing and at each storage date. The results were expressed as g 100 g−1 of initial fresh weight (FW).
Overall quality (OQ) was evaluated by an informal panel consisting of twelve people (seven men and five women, aged 24–60) using a 1 to 5 scale, in which 5 = excellent or having a freshly harvested appearance and full visual and sensory acceptability (e.g., no yellowing or browning, free from any off odors, defects, and decay), 3 = fair/still acceptable and marketable (e.g., appearance of minor defects or moderate color alteration), and 1 = poor/unmarketable, presence of off odors, extensive color changes, and major defects or decay symptoms.
Leaf color was measured on the upper side of ten randomly selected leaves (two points of photosynthetic tissue per leaf) for each sample using a colorimeter (Chroma-meter CR-400, Minolta Corp., Ltd., Osaka, Japan). The instrument recorded the CIELAB color components L* (lightness), a* (positive values for reddish colors and negative values for greenish colors), and b* (positive values for yellowish colors and negative values for bluish colors). From a* and b* values, hue angle (h°) and Chroma (C*) were calculated as h° = 180° + arctan(b*/a*) [
45] and C* = (a*
2 + b*
2)
1/2.
Twenty grams of each sample were then homogenized with H
2O (1:2 w/v), and the homogenates were centrifuged at 3500 rpm for 10 min. The supernatants were analyzed to determine total soluble solids (TSS), titratable acidity (TA), and ascorbic acid and nitrate content. TSS was determined in °Brix using a digital refractometer (MTD-045nD, Three-In-One Enterprises Co. Ltd., New Taipei City, Taiwan). TA was calculated by titrating 10 mL of extract with 0.1 M NaOH up to pH 8.1 and expressed as mg of citric acid per 100 g of fresh weight (FW). Ascorbic acid and nitrate contents (respectively, as mg 100 g
−1 and mg kg
−1 FW) were determined reflectometrically using a Reflectometer RQflex10 Reflectoquant and the Reflectoquant ascorbic acid and nitrate test strips (Merck, Darmstadt, Germany) (procedures described in art. 1.16971.0001 and 1.16981.0001 by Merck [
46]).
2.3. Statistics and Principal Component Analysis
A completely randomized design with four replicates per treatment was performed. To determine the effect of the GA3 level on yield of each leafy vegetable, a one-way ANOVA was performed (for replicated tubs for each GA3 level). Differences between means were determined by Tukey’s multiple-range test at P ≤ 5%. The effect of GA3 concentration and storage time on each leafy vegetable was determined, performing a two-way ANOVA (for replicated bags for each GA3 level and each storage time). Mean values were compared by the least significant differences (LSD) test at P ≤ 5% to identify significant differences among treatments and significant interactions between factors.
Principal component analysis (PCA) was performed on morpho-physiological and phytochemical parameters to determine the dominant parameters that were most effective in discriminating between preharvest GA3 treatments and storage time (SPSS version 13.0 (SPSS Inc., Chicago, IL, USA) statistical software). The input matrix for the analysis consisted of weight loss, TSS, TA, ascorbic acid and N-NO3− content, L*, a*, b*, Chroma, Hue angle, and overall quality (OQ). The optimum number of principal components (PCs) was assessed, retaining factors with eigenvalues higher than 1.0. The first and second principal components (PCs) were used to determine the score plot and loading matrix.
4. Discussion
Plant growth regulators (PGRs) have been used in agriculture since their discovery to control plant processes (germination, vegetative growth, reproductive development, maturity, senescence, and postharvest preservation) [
17]. Gibberellins are PGRs associated with many plant physiological activities [
47] that have been used commercially to improve morphological and phenotypic characteristics, and earliness and yield of many vegetable and ornamental crops [
48]. Gibberellins have also been tested as postharvest treatments, with the aim of preserving stored products from decay and biotic or abiotic stress. The doses of GA
3 applied to plants may vary greatly. Many experiments have been carried out to study the effects of spraying exogenous GA
3 at very low concentrations on various crops [
18,
25,
36,
37,
40,
49,
50,
51], showing that GA
3 relative concentrations and responses may differ according to species and their growth stages [
34]. This indicates the need to evaluate the effects of applying different GA
3 doses to different vegetables crop during both growth and postharvest. In fact, many crop practices may exert their effects on products also after harvesting. At harvest, vegetables are removed from the parent plant and so they lose the normal supply of water, nutrients, and organic molecules including hormones, which may be supplied by translocation from other parts of the plant during growth. Nevertheless, endogenous hormones go on functioning and controlling physiological processes of vegetables even during storage [
52]. This suggested an opportunity to control and slow postharvest changes in fruits, vegetables, and flowers by exogenous application of phytohormones. Postharvest GA
3 treatments have been used in preserving stored products because they were shown to be efficient, nontoxic, biodegradable, and with no residual [
30,
31,
32,
33]. However, little information is available concerning the role of preharvest and postharvest GA
3 treatments during cold storage of minimally processed leafy vegetables. Thus, we aimed to determine if the preharvest application of GA
3 through the MNS of a hydroponic floating system could affect quality characteristics of minimally processed leaf lettuce and rocket at harvest and during cold storage.
The modification that plant growth regulators can cause on plant phenotype, growth, and development by altering the hormonal content and their balance may play a role in modifying crop yield [
53,
54,
55,
56,
57]. Leaf lettuce and rocket yield were positively affected by exogenous GA
3 supplied through the MNS at 10
−6 M GA
3 [
42]. The growth-promoting effect of GA
3 resulted in a significantly higher yield of both raw and minimally processed leafy vegetables. GA
3-treated plants showed an enhanced activity of enzymes that have a role in photosynthetic processes [
58,
59]; thus, GA
3 may indirectly increase the photosynthetic rate [
52,
54,
58,
60], leading to greater dry mass accumulation and higher yield.
Weight loss occurring during storage is one of the main issues that may negatively influence the appearance and quality of minimally processed leafy vegetables. It could be directly linked with water loss, but also with respiration that degrades carbohydrate reserves [
61,
62]. This parameter may be used as an index of the impact of abiotic stress and storage conditions on fresh cut products [
63,
64]. Minimally processed vegetables are usually packed in sealed plastic bags that have low permeability to water vapor; this characteristic determines the quick rise in relative humidity (RH) to a very high level (almost 100% RH) inside the bags [
65,
66,
67,
68], so dehydration is not a main issue, as we found for the leafy vegetables tested in this work. The weight losses of minimally processed lettuce and rocket leaves were similar on average at the end of storage; GA
3 preharvest application was effective in reducing weight loss of rocket leaves but had no effect on lettuce leaves. Water loss is mainly due to the reduction of the resistance of outer periderm or cuticle to transpiration, caused by processing or by leaf tissue deterioration [
69]. GA
3 may influence cuticle thickness and epidermal cell dimension, but to different extents based on species and cultivar [
70], as we found for leaf lettuce and rocket, and may also have a role in decreasing the activity of cell wall-hydrolyzing enzymes [
71]. Even though plant cell walls are permeable to water, the higher integrity of this structure might increase resistance to water flux [
72]. Thus, the lower weight loss observed in the GA
3-treated rocket leaves might be attributed to the maintenance of tissue integrity due to lower activity of enzymes responsible for decomposing cellular structure [
71].
Total soluble solids and titratable acidity were not affected by GA
3 preharvest supply in both lettuce and rocket but showed variations during cold storage. Minimally processed leaves may increase their respiration activity and ethylene production during storage, and these metabolic variations could influence the amount of complex and soluble sugars and organic acids. Thus, the increase of TSS and total acidity found after 7 and 14 d of storage could be an indirect indication of the increasing metabolic activity of leaf lettuce and rocket leaves during cold storage. Similar increases of total acidity have been found during cold storage of minimally processed cauliflower [
68,
73], fresh-cut red chicory [
65], and escarole [
66]. Soluble sugars are transformed to organic acids and to other simple carbon compounds, thus determining the contemporary increase of titratable acidity during storage in both leafy vegetables. Lers et al. [
74] found that preharvest GA
3 treatments were effective in reducing the respiration rate of parsley leaves stored for 8 d at 25 °C, while in our experiment we did not find a modification of metabolites (soluble sugars or total acidity) that could have confirmed the effect of GA
3 on respiration rate. This was probably due to the low temperature and to the atmosphere inside the sealed bags that was effective in slowing the respiration process [
65,
75].
Among the organic acids found in vegetables, ascorbic acid (vitamin C) is an important element of nutritional quality, as its consumption has been related to a lower incidence of several chronic diseases including cardiovascular disease and cancer [
76]. Producing vegetables with a high vitamin C content and maintaining the level of vitamin C during postharvest storage time is essential for enhancing their nutritional value. Leaf lettuce had a lower ascorbic acid content than rocket and was not influenced by GA
3 treatment or cold storage. Even if ascorbic acid is a very labile compound that can be quickly degraded during postharvest storage of perishable foods, several studies have shown that its losses can be strongly limited or that it can even increase in packed cold-stored vegetables [
67,
77,
78,
79,
80]. In fact, in our work, the content of ascorbic acid of rocket was positively affected by cold storage. Furthermore, growing rocket with 10
−6 M GA
3 in the MNS resulted in a higher content of ascorbic acid in rocket leaves at harvest and during cold storage. A similar effect on vitamin C content was found by El-Hamahmy et al. [
81] on sugar snap peas after postharvest treatment with GA
3 and during cold storage. The different response of leaf lettuce and rocket as regards ascorbic acid content could be related to differences in metabolic processes between the tested species. A relationship between the ascorbic acid content of leaves and nitrogen availability has been found in some fruits and vegetables, but this relationship may vary depending on many factors (e.g., genus, climate, etc.) [
82,
83]. Leafy vegetables need sufficient N supply to maintain ascorbic acid synthesis [
67,
84]. GA
3 treatments can affect nitrogen metabolism and nitrogen redistribution to different plant organs and to different biochemical processes [
42], thus, likely influencing ascorbic acid production.
The differing effect of GA
3 on leaf lettuce and rocket was also found for their nitrate content. Nitrate accumulation in leafy vegetables can negatively affect human health and determine a decrease of nutritional quality or even the loss of marketability [
85]. Minimally processed lettuce leaves showed an average initial nitrate content of 2448.3 mg kg
−1 FW that decreased during cold storage in GA
3-treated leaves, while it remained constant in control leaves. Rocket leaves also exhibited a decrease of nitrate content during storage, but irrespective of GA
3 treatments which, in turn, significantly affected initial nitrate accumulation, with a lower value with 10
−6 M GA
3 treatment. Gibberellic acid affects nitrogen metabolism and nitrogen redistribution in plants and has a positive effect on nitrate reductase activity [
58,
86]. Thus, GA
3 treatments, above all 10
−6 M GA
3, might have influenced nitrate reductase, but in different ways in the tested leafy vegetables. The increased activity of this enzyme could have caused a reduction of nitrate accumulation in rocket during plant growth, while leaf lettuce plants may not have reduced the nitrate content during growth, for a more efficient translocation from roots to leaves and, when leaves were detached and deprived of nitrate supply, could have finally showed the higher nitrate reductase efficiency, promoted by GA
3 treatment.
The quality of vegetable products, and those minimally processed above all, is strongly characterized by appearance and color. These characteristics can impact food choice and acceptability and may also influence the consumer’s perception of sensory quality. Color modifications may be triggered by preharvest [
42,
67,
87] or postharvest [
65,
66,
68,
88] factors. Color alterations that may occur in leafy vegetables are mainly related to browning and yellowing (chlorophyll degradation) and could determine marketability loss before physical, chemical, and microbiological alterations are perceivable [
67]. During storage, color modifications were noted in lettuce and rocket at different extents for each color parameter. The effect of preharvest GA
3 treatment was more significant for rocket than for lettuce. Nonetheless, at the end of the cold storage period, GA
3-treated leaves of both leafy vegetables showed a higher hue angle that corresponds to a greener color and consequently to a higher chlorophyll content [
89,
90]. Preharvest [
74] or postharvest [
30,
91] GA
3 treatments have been effective in retarding various senescence processes (e.g., chlorophyll and protein loss and amino acid accumulation) in other leafy vegetables. The degradation of chlorophyll in detached leaves is attributed to the lack of sufficient endogenous hormones, which cease on excision of the leaves from the plant [
82,
92]. The inhibitory role of GAs in chlorophyll degradation has been confirmed in some other plant species [
81,
93,
94,
95,
96].
Color retention during cold storage influences quality perception of the minimally processed leafy vegetable tested. Even small variations of color components can result in significant changes of color that can be noted by the human eye, as confirmed by the panel that assessed the overall quality. Scores for lettuce OQ decreased during storage, with significantly higher values than the controls at the end of storage, following 10
−6 M GA
3 treatment. Cold storage in sealed plastic bags was effective in retarding alteration in appearance for at least two weeks [
65,
66,
68,
73,
75,
97]. Moreover, the slowed senescence of GA
3-treated samples maintained an overall visual quality over the threshold of marketability in both lettuce and rocket for up to 21 d of cold storage.
As seen above, minimally processed leaf lettuce and rocket were in some cases differently affected by GA
3 preharvest treatments. This difference is clearly shown by the Principal Component Analysis that summarize the different responses of the two leafy vegetables to preharvest treatments. Postharvest responses to preharvest GA
3 treatments were species-specific and dose-dependent, thus confirming that hormone requirement, relative concentrations, and responses may vary for different species [
34].